Using both cell culture and in vivo models of infection, our collaborative group investigates the molecular pathogenesis of positive strand RNA viruses of global importance. West Nile virus (WNV) is an emerging mosquito-borne human and animal pathogen that has caused outbreaks of fatal encephalitis in Europe, Asia, the Middle East, and most recently, the United States. At present, there is no available antiviral therapy or vaccine. This proposal is directed at the discovery of agents that inhibit replication of WNV with the long-term goal of developing antiviral agents to treat WNV-associated disease. Several independent approaches are used to identify inhibitory agents against WNV and the degree of their antiviral effects. One goal of this grant is to develop nucleic-acid specific antagonists against WNV by generating small interfering double-stranded RNA based on the recently described phenomenon of RNA interference (Specific Aims 1 and 2). These antagonists will be tested in cells and in vivo in a murine model of West Nile encephalitis. A second goal is to use genetic screens to identify the viral determinants that mediate sensitivity and resistance to RNA interference (Specific Aim 3). The third goal is to utilize subgenomic WNV replicons as a screening tool to identify small molecule inhibitors of WNV replication (Specific Aim 4). The identification of sequence-specific and small molecule antagonists of WNV and the characterization of pharmacologic enhancers and/or suppressors of RNA interference will provide a platform for the development of novel therapeutic agents that may be used against WNV as well as other positive strand RNA viruses.